Abstracts of the - Med Uni Graz ESCHERICH SYMPOSIUM 2016: ABSTRACTS Table of Contents 4 P14 Effects of probiotic therapy on the microbiota and mucosal immune system in patients with

Abstracts of the

3rd Theodor Escherich Symposium

on Medical Microbiome Research

3rd - 4th November 2016

Graz, Austria

THEODOR ESCHERICH SYMPOSIUM 2016: ABSTRACTS

Table of Contents

2

Short Talks

T1

Changes of the colonic microenvironment in multiple sclerosis

Adrian Mathias Moser, Walter Spindelboeck, Heimo Strohmaier, Christian Enzinger, Thomas Gattringer, Siegrid Fuchs, Franz Fazekas, Gregor Gorkiewicz, Philipp Wurm, Christoph Högenauer, Michael Khalil

5

T2 The altered microbiome of confined built environments

Alexander Mahnert, Christine Moissl-Eichinger, Markus Zojer, Thomas Rattei and Gabriele Berg 6

T3 Extremophily vs. pathogenicity: New insights into the genus Yersinia

Alexandra Perras, Martin Grube, Gabriele Berg, Ivo Steinmetz, Robert Krause, Christine Moissl-Eichinger and the MASE team

7

T4

Probiotic modulation of gut microflora in cirrhosis: Influence on Lactobacilli and Veillonella

A. Horvath, M. Bashir, B. Leber, B. Schmerboeck, M. Tawdrous, F. Rainer, P. Douschan, E. Krones, W. Spindelboeck, F. Durchschein, G. Zollner, RE. Stauber, P. Fickert, P. Stiegler, V. Stadlbauer

8

T5 Critical issues in mycobiota analysis

Bettina Halwachs, Nandhitha Madhusudhan, Robert Krause, R. Henrik Nilsson, Christine Moissl-Eichinger, Christoph Högenauer, Gerhard G. Thallinger, Gregor Gorkiewicz

9

T6 The microbiome of Brassicaceae and its benefits for plant and human health

Birgit Wassermann, Daria Rybakova, Gabriele Berg 9

T7

Exploring distal-gut microbial ecology and host-microbe metabolic interactions by in vivo stable isotope probing with 13C-glucose ureide

Buck T. Hanson, Orest Kuzyk, Thomas Decker, Andreas Richter, Wolfgang Wanek, Douglas Morrison, David Berry, Alexander Loy

10

T8 The correspondence of nasal microbiome and normal olfactory function

Kaisa Koskinen, Johanna Reichert, Christine Moissl-Eichinger, Jochen Schachenreiter, Stefan Hoier, Veronika Schöpf

11

T9

A Donor microbiota signature correlating to clinical efficacy in repeated faecal microbiota transplantation in therapy refractory ulcerative colitis

Wurm P, Kump PK, Gröchenig HP, Wenzl HH, Petritsch W, Halwachs B, Wagner M, Stadlbauer-Köllner V, Eherer AJ, Hoffmann KM, Deutschmann A, Reicht G, Reiter L, Slawitsch P, Gorkiewicz G, Högenauer C

12

T10 Functional and structural characterization of a Gram positive Type IV Secretion System

Verena Kohler, Andreas Aufschnaiter, Sabrina Büttner, Rebekka Biedendieck, Elisabeth Grohmann and Walter Keller

13


Table of Contents

3

Poster Presentations

P1 The influence of PMA on the microbial diversity on stool samples

Alexandra Perras, Lisa Wink, Michael Beck, Maximilian Mora and Christine Moissl-Eichinger 14

P2 Human age and skin physiology shape diversity and abundance of Archaea on skin

Christine Moissl-Eichinger, Kaisa Koskinen, Peter Wolf 14

P3 Pilot study of the metabolisation of an Arctic root extract by gut microbiota

Ivana Turek, Kaisa Koskinen, Christine Moissl-Eichinger, Franz Bucar 15

P4 Optimization of Archaea detection in human samples

Manuela-Raluca Pausan, Alexandra K. Perras, Kaisa Koskinen, Michael Beck, Maximilian Mora, Robert Krause, Corinna Bang, Ruth Schmitz-Streit, Christine Moissl-Eichinger

16

P5 Assessing the resistance potential of microorganisms from archived samples of the International Space Station

Maximilian Mora, Alexandra Perras, Tatiana Alekhova, Robert Krause, Christine Moissl-Eichinger

17

P6 Expression and Interaction Studies of the Transfer Proteins TraN and TraK from the Gram-positive Conjugative Model Plasmid pIP501

Melanie Müller, Verena Kohler, Walter Keller

18

P7

Experimental evolution of polysaccharide-degrading Bacteroides thetaiotaomicron, an abundant member of the human gut

Nika Ivanovova, Christos Zioutis, Fatima Pereira, Madeleine Wyss, Andrew J. Macpherson, Kathy D. McCoy, David Berry

19

P8 Antimicrobial volatile organic compounds: Identification of intra-cellular targets

Pascal Mülner, Laura Lange, Tomislav Cernava, Stefan Liebminger, Sepp D. Kohlwein, and Gabriele Berg 20

P9 Community changes during the decontamination of hatching eggs using diazine derivates

Peter Kusstatscher, Tomislav Cernava, Stefan Liebminger, Gabriele Berg 20

P10 Genus Lactobacillus and subcutaneous adipose tissue thickness in women

Mörkl, S., Lackner, S., Müller, W., Gorkiewicz, G., Kashofer, K., Oberascher, A., Amouzadeh-Ghadikolai, O., Holasek, S.J.

21

P11 Polyesterases from moss-assotiated microorganisms

Veronika Perz, Felice Quartinello, Christoph Provasnek, Georg Guebitz, Gabriele Berg, Christina A. Mueller 22

P12 Effect of UV-R on skin microbiome and its interference in UV-induced immune suppression

VijayKumar Patra, Bettina Halwachs, Gregor Gorkiewicz and Peter Wolf 23

P13 Investigation of the possible link between genetic background, gut microbiota and metabolism using the AJ/C57 chromosome substitution strains (CSSs)

Zahra. Safari, Karl. Kashofer, Christine. Moissl-Eichinger, Gerald. Höfler, Kurt. Zatloukal

24


Table of Contents

4

P14

Effects of probiotic therapy on the microbiota and mucosal immune system in patients with diarrhea-predominant irritable bowel syndrome

Adrian Mathias Moser, Bettina Halwachs, Walter Spindelboeck, Heimo Strohmaier, Patrizia Kump, Gregor Gorkiewicz, Christoph Hoegenauer

25

P15 Adaptation of Escherichia coli and Staphylococcus aureus to the natural compounds carvacrol and p-cymene and its impact on their antimicrobial susceptibility

Šimunović K., Klanćnik A., Janosity A., Tušek Žnidari? M., Bucar F., Kisko G., Smole Možina S.

26


SHORT TALKS

5

T1 Changes of the colonic microenvironment in multiple sclerosis

Adrian Mathias Moser1,2*

, Walter Spindelboeck1,2*

, Heimo Strohmaier3, Christian Enzinger

4, Thomas Gattringer

4,

Siegrid Fuchs4, Franz Fazekas

4, Gregor Gorkiewicz

2,5, Philipp Wurm

2,5, Christoph Högenauer

1,2, Michael Khalil

4

1 Department of Internal Medicine, Division of Gastroenterology and Hepatology, Medical University of Graz, Auenbruggerplatz 15, 8036 Graz, Austria

2 Theodor Escherich Laboratory for Microbiome Research, Medical University of Graz, Auenbruggerplatz 15, 8036 Graz, Austria

3 Center for Medical Research, Medical University of Graz, Stiftingtalstraße 24, 8010 Graz, Austria

4 Department of Neurology, Medical University of Graz, Auenbruggerplatz 22, 8036 Graz, Austria

5 Institute of Pathology, Medical University of Graz, Auenbruggerplatz 25, 8036 Graz, Austria

Background: The understanding of multiple sclerosis (MS), a disabling autoimmune disease of the central nervous system, is currently challenged by the possible role of the gastrointestinal tract in neuroinflammation. This comes from a mounting body of evidence showing the effects of an altered microbiome on intestinal barrier and mucosal immune function from animal studies. However, human data remain to be scarce. Methods: We analysed dendritic cell (DC) and T cell subsets using FACS analysis of colonic mucosal specimens of the left- and right-sided colon in 15 MS patients and 10 healthy controls. Specimens were obtained during colonoscopy. GC-MS measurements of stool samples measuring short-chain fatty acids (SCFA) content and 16s rDNA analysis were performed in fecal samples. Results: FACS analysis of colonic mucosal specimens showed decreased numbers of total dendritic cells (DC), CD103+ tolerogenic DCs and CD4+CD25+127- regulatory T-cells (Tregs) in the left-sided but not in the right-sided colon of MS patients. GC-MS measurements of fecal samples revealed lower levels of short-chain fatty acids (SCFAs, especially butyrate and acetate) in stools of MS patients. In addition, 16S rDNA analysis of fecal samples showed changes of bacterial strains predominantly belonging to the Clostridiales order. Conclusion: Our findings indicate a disturbed homeostasis of colonic DCs and Tregs in MS patients. This may be associated to colonic SCFA depletion triggered by specific microbial alterations. Further research could elucidate the role of the colonic SCFA profile or the colonic Treg pool in modifying the course of MS


SHORT TALKS

6

T2 The altered microbiome of confined built environments

Alexander Mahnert, Christine Moissl-Eichinger, Markus Zojer, Thomas Rattei and Gabriele Berg Institute of Environmental Biotechnology, Graz University of Technology, Graz, Austria Understanding the complex influences of human maintenance, environmental confinement and microbial control in built environments is critical to increase our health inside buildings. However, to date the scientific community lacks clear concepts how to improve our well-being inside buildings. For conceiving such an improved concept, we sampled different built environments, characterized by an increasing level of microbial control, (public buildings, public and private houses, intensive care units, cleanroom facilities) in a large scale, to evaluate associated structural and functional changes of the microbiome. Genome centric shotgun metagenomics were applied to investigate functional capabilities of similar species from different built environments. Both opposing environments could not only be characterized by discriminant profiles of its species (Archaea, viruses or arthropods inside confined built environments), but also by numerous functional features. According to this analysis, uncontrolled built environments were populated by gram-positive bacteria with functions associated to carbohydrate and amino acid metabolism, while controlled built environments were characterized by gram-negative bacteria with many functions associated to virulence, disease and defense. This trend was also visible on the level of binned genomes. Hence, genomes closely related to Acinetobacter and derived from all sampled built environments revealed a higher proportion of genes coding for virulence, disease, resistance or defense in microbial controlled built environments. This study indicates which conditions enforce a microbiome to develop adverse functional capabilities and might serve as a model for attempts to better design and control (biotechnologically) microbiomes indoors in the future for human health.


SHORT TALKS

7

T3 Extremophily vs. pathogenicity: New insights into the genus Yersinia

Alexandra Perras, Martin Grube, Gabriele Berg, Ivo Steinmetz, Robert Krause, Christine Moissl-Eichinger and the MASE team Medical University of Graz, Graz, Austria In the frame of the MASE project ((Mars Analogues for Space Exploration, http://mase.esf.org/), an astrobiological and medical relevant microorganism was isolated from an extreme, Mars-analog habitat: Yersinia sp LG1. Yersinia is a bacterial genus in the family Enterobacteriaceae with known pathogenic representatives, such as Y. pestis. Y. pestis is the causative agent of the Great Plague and, whilst being a tremendous threat for the well-being, rather known to be highly adapted to (moderate) conditions prevalent in human beings. Although the known Yersinia species (including Y. pestis) have not been described to possess extraordinary resistance properties, , Yersinia sp LG1 revealed a surprisingly and not expected spectrum of adaptations to extreme stressors such as desiccation and ionizing radiation, whilst lacking pathogenesis capacities. Interestingly, many publications report the detection of (opportunistic) pathogenic microorganisms (or close relatives thereof) in environmental settings, including extreme environments. We suggest that the process of “breaking bad” is either associated to evolutionary processes, or triggered by the transfer from one (extreme) habitat to another (e.g. the human body). In the light of the antibiotic crisis, an urgent effort is needed to shed light into the trigger of this process and to prevent it. Consequently, we want to draw attention to these inter-habitat connections.


SHORT TALKS

8

T4 Probiotic modulation of gut microflora in cirrhosis: Influence on Lactobacilli and Veillonella

A. Horvath, M. Bashir, B. Leber, B. Schmerboeck, M. Tawdrous, F. Rainer, P. Douschan, E. Krones, W. Spindelboeck, F. Durchschein, G. Zollner, RE. Stauber, P. Fickert, P. Stiegler, V. Stadlbauer Medical Univerity of Graz, Department of Gastroenterology and Hepatology, Graz, Austria Liver cirrhosis is associated with profound changes in the gut microflora. In this study we aimed to characterize these changes in stable cirrhotic patients (Child-Pugh score < 11) and assess the modulating effects of six months probiotic intervention with Ecologic Barrier®, also known as Omnibiotic Hetox®, (Winclove, The Netherlands) in a randomized, double-blind, placebo-controlled trial. Bacteria were identified by next generation sequencing of hypervariable regions of 16S rDNA obtained from stool samples. We found that in 87 stable cirrhotic patients alpha diversity was significantly reduced compared to 29 controls (chao1 index: 1613±570 and 2723±890, respectively, p=0.001). In accordance to this decrease, unclassified bacteria were significantly decreased in cirrhotic patients compared to controls (p=0.001). In addition, 26 genera were differentially abundant between cirrhotics and healthy controls, including Veillonella (p=0.001), Lactobacillus (p=0.001), Blautia (p=0.001) and Streptococcus (p=0.002). Six months of probiotic intervention with a multispecies probiotic (n=44) or placebo (n=36) significantly increased the abundance of a Lactobacillus species (p=0.041) and tended to decrease the abundance of Veillonella (ns). Both effects were reversed six months after the intervention had stopped. Probiotic intervention did not affect alpha diversity. In conclusion, we found a substantial overrepresentation of Veillonella in cirrhotic patients, an LPS producing opportunistic pathogen mostly found in the oral cavity. Probiotic supplementation raised the abundance of Lactobacillus and reduced the abundance of Veillonella during the intervention. The alterations were transient and returned to baseline conditions six months after the end of treatment.


SHORT TALKS

9

T5 Critical issues in mycobiota analysis

Bettina Halwachs, Nandhitha Madhusudhan, Robert Krause, R. Henrik Nilsson, Christine Moissl-Eichinger, Christoph Högenauer, Gerhard G. Thallinger, Gregor Gorkiewicz Medical University of Graz, Institute of Pathology, Graz, Austria The development of high-throughput molecular methods has revolutionized our understanding of the microbiota throughout the last years. By now, microbiota research was mainly focused on the bacterial component of the microbial community. But it comprise apart from bacteria and archaea also fungi, lichens, phages, and even viruses. Fungi represent the second largest kingdom of eukaryotic life and hundreds of fungal species are regularly involved in human and animal mycoses, such as Aspergillosis, Candidiasis, or Mucormycosis. However, the tools used for microbial community analysis are typically developed for the bacterial component (16S rRNA amplicon surveys). Hence they often fall short or are even not appropriate, if applied for fungal marker-gene investigations. Here we guide through all major steps of a typical fungal amplicon dataset analyses with a special focus on the bioinformatical challenge of otu-picking strategies in fungal datasets. At the example of a fungal ITS in silico mock community we reveal common pitfalls within the popular analysis workflows in combination with solutions on how to overcome these problems.

T6 The microbiome of Brassicaceae and its benefits for plant and human health

Birgit Wassermann, Daria Rybakova, Gabriele Berg

Graz University of Technology, Environmental Biotechnology, Graz, Austria In recent years, the microbiome has been identified as a key factor for plant health. The microbiota inhabiting edible plants can also have a meaning for human health. The microbiome of the edible parts of seven Brassica vegetables, each purchased at the farmer´s market and the supermarket, was examined for beneficial effects for human health and for putative biocontrol agents. The hydrolysis of glucosinolates by myrosinase forms isothiocyantes which trigger a host defence system in mammals protecting them against cancer. Due to the fact that the plant myrosinase is inactivated by storage and cooking, it is of interest to identify vegetable-associated microbiota with myrosinase activity, for improving health benefits of Brassica vegetables. Four different approaches to determine bacterial myrosinase activity were carried out: i) GOD-POD coupled enzyme assay; ii) HPLC; iii) selective agar plates and iv) degenerate primers for a selective amplification of a myrosinase gene. The experiments confirmed myrosinase activity for Enterobacter cloacae, the putative myrosinase gene was identified. Evaluating the influence of the point of sale demonstrated that the composition of the microbial community is rather driven by the type of vegetable host than by the purchase source. Significant cultivar-specific influence of the purchase origin was determined. Four strains, isolated from farmer´s market vegetables, were identified to combat plant pathogen Verticillium longisporum and to reveal potential plant growth promoting properties. Overall, the data show a high microbial diversity in different Brassica vegetables that has a meaning for both the plant health as well as to human health.


SHORT TALKS

10

T7 Exploring distal-gut microbial ecology and host-microbe metabolic interactions by in vivo stable isotope probing with 13C-glucose ureide

Buck T. Hanson, Orest Kuzyk, Thomas Decker, Andreas Richter, Wolfgang Wanek, Douglas Morrison, David Berry, Alexander Loy University of Vienna, Division of Microbial Ecology, Vienna, Austria Microbial metabolites produced from the fermentation of complex polysaccharides in the distal-gut have far-reaching, often beneficial effects on host physiology. In contrast, dietary overabundance of simple sugars such as glucose can have harmful effects on host health and energy homeostasis. To explore the influence of dietary glucose on distal gut microbial ecology, we have implemented 13C-glucose ureide as a model substrate for colon-specific delivery of isotopically-labeled glucose. When wild-type mice were orally dosed with 13C-glucose ureide, we observed respiration of 13CO2, isotopic labeling of cecal-derived biomass and DNA, in addition to the detection of 13C-labeled microbial-derived metabolites (acetate, propionate, and butyrate) in host blood plasma. The peak time of compound metabolism was associated with increased abundance of 16S rRNA gene phylotypes related to glucose-fermenting members of the Lachnospiraceae and Erysipelotrichaceae that are associated with obesity. To examine the role of dietary-induced shifts in gut microbiota and the fate of colonic glucose, we provided mice with two isocaloric diets: a control starch-based diet or starch-free glucose-rich diet. After three weeks with no significant differences in weight gain, each mouse received an oral dose of 13C-lableled glucose ureide. Using a combination of mass spectroscopy, microscopic, and biomarker analyses, we currently aim to connect shifts in microbial community structure and physiology with the fate of glucose-derived metabolites and contributions to host physiology.


SHORT TALKS

11

T8 The correspondence of nasal microbiome and normal olfactory function

Kaisa Koskinen, Johanna Reichert, Christine Moissl-Eichinger, Jochen Schachenreiter, Stefan Hoier, Veronika Schöpf Medical University of Graz, Graz, Austria The human body is populated by trillions of microorganisms with tremendous effect on our health, disease and behaviour. The microbiome is like a personal fingerprint, and besides the key- and core microbes, our microbial partners are individual. Human microbiome changes during the course of life, and in particular reduced diversity causes a variety of health problems.

The present study aimed to explore the association between the structure of the nasal microbiome and the smelling ability of healthy human volunteers. On the same day, we first assessed olfactory function with the TDI test using the SniffinSticks battery, and later olfactory mucosa microbiome samples were taken from the same study subjects using nasal swabs. Microbial community structure and diversity were examined by 16S rRNA sequencing using Illumina MiSeq.

The first results with 49 normosmic, healthy subjects suggest a relation between archaeal genus Methanobrevibacter and overall olfactory function (total TDI). Interestingly, study subjects with high abundance of Methanobrevibacter had never very good sense of smell. Specifically in female study subjects, the abundance of Methanobrevibacter seemed to correlate negatively with olfactory function. Additionally, we found correlations between certain bacterial genera and odour threshold, discrimination and identification.

This study will provide novel knowledge on the nasal microbiome and its potential application as an effective biomarker for smelling function, and possibly generate new ideas for therapeutic solutions. In this presentation, we will summarise the latest results of this project.


SHORT TALKS

12

T9 A Donor microbiota signature correlating to clinical efficacy in repeated faecal microbiota transplantation in therapy refractory ulcerative colitis

Wurm P, Kump PK, Gröchenig HP, Wenzl HH, Petritsch W, Halwachs B, Wagner M, Stadlbauer-Köllner V, Eherer AJ, Hoffmann KM, Deutschmann A, Reicht G, Reiter L, Slawitsch P, Gorkiewicz G, Högenauer C Medical University of Graz, Institute of Pathology, Graz, Austria Introduction: Fecal microbiota transplantation (FMT) are investigated in various studies as an alternative approach for treatment of ulcerative colitis (UC). Nevertheless the results of this studies show contrary results and varying in response rates to FMT. Additionally factors influencing the response in FMT in UC is still unknown. Aims and Methods: Our aim was to investigate the microbiota of UC patients treated with FMT and their respective donors in a prospective trial to define a microbial signature that correlates to clinical efficacy of FMT. 17 patients were treated with repeated FMT (n=5) every 14 days after an antibiotic pre-treatment for 10 days with FMTs from the same donor. The assesement of clinical efficacy was determined by the total Mayo score at day 90. Patients were classified as responders by a reduction in total Mayo score of at least 3 points, and remission showing a total Mayo score less or equal 2 points to this treatment. Results: 59% (10/17) of all patients showed a response and 24% (4/17) even a remission to FMT. A successful donor microbiota engraftement was not related to treatment response. Furthermore the donor microbiota correlating to remission showed an increased microbial richness, a significant separation from the donor microbiota inducing no response based on the taxonomic and phylogenetic composition and higher levels of Akkermansia muciniphila and Ruminococcus spp. Conclusion: The signature of the donor microbiota is a major factor for efficacy of FMT in chronic active UC. Therefore selection of the donor by screening the microbiota might increase efficacy of FMT in chronic active UC.


SHORT TALKS

13

T10 Functional and structural characterization of a Gram positive Type IV Secretion System

Verena Kohler, Andreas Aufschnaiter, Sabrina Büttner, Rebekka Biedendieck, Elisabeth Grohmann and Walter Keller University of Graz, Institute of Molecular Biosciences, Graz, Austria Spreading of antibiotic resistances in bacteria is an enormous issue nowadays, causing tremendous problems in healthcare, and Gram positive (G+) pathogens are among those most responsible for nosocomial infections. However, scarce information about systems responsible for dissemination of drug-resistances in G+ is available. Based on its prevalence in numerous bacterial pathogens, we are studying the conjugative model plasmid pIP501, coding for 15 transfer-proteins (tra-proteins) of a simplified type IV secretion system (T4SS). With the final goal of gaining novel information regarding the transfer machinery of our T4SS, we focus on structural and biochemical characterization of the membrane-inserted core complex, responsible for the actual transfer of the ss-DNA during conjugation. Utilizing a bacterial-2-hybrid system (BACTH), we identified interacting key players of the membrane-resident complex of pIP501. With extremely well titratable polycistronic expression in Bacillus megaterium, a novel G+ protein production host, we aim for assembly and membrane integration of this core complex in the correct stoichiometry. First trials revealed reasonable expression levels of the recombinant tra-proteins. Bacillus megaterium, beyond its excellent suitability for recombinant expression of our G+ T4S system, harbors additional features, enabling us to perform localization studies with immunocytochemistry as well as interaction experiments using in vivo crosslinking approaches. In addition to X-ray crystallography, the structural characterization of this protein complex will be done with Cryo-electron microscopy methods.


POSTER PRESENTATIONS

14

P1 The influence of PMA on the microbial diversity on stool samples

Alexandra Perras, Lisa Wink, Michael Beck, Maximilian Mora and Christine Moissl-Eichinger Medical University of Graz, Graz, Austria Disease patterns are often caused by a dysbiosis of the healthy microbiota in the human gut. The investigation of the microbial community of stool samples is a first step to elucidate potential pathogenic agents as well as missing members of the healthy flora. However, conventional next generation sequencing methods lack the opportunity to differentiate between the viable and dead microbial proportion. The chemical compound propidium monoazid (PMA) when added to samples,masks free DNA of those cells, which have a disrupted cell wall (i.e. dead cells). After the exposure to light, which completes the reaction, the whole DNA fraction can be extracted, introduced to PCR reactions and the viable proportion can be visualized. This technique is already standardized in food safety monitoring and environmental microbiology, however, is barely used in clinical studies and a sample-specific protocol is not available yet. The key step of the PMA reaction is highly dependent on the turbidity of samples, as light has to transmit the entire sample. This can be resolved by adjusting the applied concentration of PMA. In this study, we aimed to determine the concentration of PMA, which shows a significant effect on the microbial community. We treated stool samples with a concentration series (0-300 µM PMA) and subjected the samples to next generation sequencing. In conclusion, a concentration of 300µM PMA is necessary to observe an effect on the microbial community composition. This work provides a standardized protocol for the application of PMA in clinical samples with a high turbidity.

P2 Human age and skin physiology shape diversity and abundance of Archaea on skin

Christine Moissl-Eichinger, Kaisa Koskinen, Peter Wolf Medical University of Graz, Graz, Austria The human skin microbiome acts as an important barrier protecting our body from pathogens and other environmental influences. Recent investigations have provided evidence that Archaea are a constant but highly variable component of the human skin microbiome, yet factors that determine their abundance changes are unknown. Here, we tested the hypothesis that the abundance of Archaea on human skin is influenced by human age and skin physiology by using quantitative PCR of 51 different skin samples taken from human subjects of various age. Our results reveal that Archaea are more abundant in human subjects either older than 60 years or younger than 12 years as compared to middle-aged human subjects. These results, together with results obtained from spectroscopy analysis, allowed us to link lower sebum levels and lipid content and thus reduced skin moisture with an increase in archaeal signatures. Amplicon sequencing of selected samples revealed the prevalence of specific eury- and mainly thaumarchaeal taxa, represented by a core archaeome of the human skin.



15

P3 Pilot study of the metabolisation of an Arctic root extract by gut microbiota

Ivana Turek1, Kaisa Koskinen2, Christine Moissl-Eichinger2, Franz Bucar1 1 Institute of Pharmaceutical Sciences, Department of Pharmacognosy, University of Graz,

Universitätsplatz 4/1, 8010 Graz 2 Department of Internal Medicine, Section of Infectious Diseases and Tropical Medicine, Medical

University Graz, Auenbruggerplatz 15, 8036 Graz, Austria Arctic root (Rose root), the roots and rhizomes of Sedum roseum (L.) Scop. (syn. Rhodiola rosea L.), have been traditionally used for relief of mental and physical symptoms of stress, such as fatigue, weakness, exhaustion, irritability and slight anxiety (HMPC, 2012). It grows mainly at the arctic regions of Europe, North America or Asia (Cuerrier et. al. 2015). Currently the phenylethanol glycoside salidroside as well as the cinnamic alcohol derivatives - the rosavins - are regarded as quality determining compounds (HMPC, 2012). As these glycosides most likely act as prodrugs, a metabolic study of a 60% ethanolic arctic root extract was performed. The dried extract was incubated with freshly harvested human gut microbiota in an anaerobic chamber taking samples after 0, 4 and 24 hours. The samples were analysed using UHPLC-PDA-HRMS analysis (Q Exactive, Thermo Scientific). The preliminary results showed that some of the main compounds like lotaustralin, rosavin, rosarin, which were present at the beginning of incubation (time point 0), were not present in samples at 4 and 24 hrs, respectively. Interestingly, cinnamyl alcohol was increasing within 4 hours, however present at lower amounts after 24 hours which indicated further metabolisation. On the other hand the microbial community was analyzed through next-generation sequencing-based microbial community analysis (on 16S rRNA gene level). It could be observed that after 24 hrs the changes in community composition affected mainly Bacteroidales (S24-7-296340; Bacteroides uniformis), Ruminococcaceae (359950, Ruminococcus), Lachnospiraceae (Coprococcus), and Enterococcaceae (Enterococcus). The preliminary results have provided insights into the interplay of plant extract and colon microbial community.

Acknowledgements: We would like to thank Mag. Dietmar Vogt, Klagenfurt, for collecting Arctic root and the “Amt

der Kärntner Landesregierung” for permitting collection of plant material. Dr. Eva-Maria Pferschy-Wenzig, Institute

of Pharmaceutical Sciences, is gratefully acknowledged for help with HR-ESI-MS analysis at the NAWI Graz Central

Lab Environmental, Plant & Microbial Metabolomics. References: HMPC, Committee on Herbal Medicinal Products,

European Medicines Agency, 2012. Assessment report on Rhodiola rosea L., rhizoma et radix.

EMA/HMPC/232100/2011, published 27 March 2012. Cuerrier A., K. Ampong-Nyarko, 2015. Rhodiola rosea in

Traditional Herbal Medicines for Modern Times. CRC Press, Taylor & Francis Group.



16

P4 Optimization of Archaea detection in human samples

Manuela-Raluca Pausan, Alexandra K. Perras, Kaisa Koskinen, Michael Beck, Maximilian Mora, Robert Krause, Corinna Bang, Ruth Schmitz-Streit, Christine Moissl-Eichinger Medical University of Graz, Graz, Austria Trillions of microbes live in and on the human body, forming the human microbiota. These complex communities contain taxa from all three domains of life (Bacteria, Eukarya and Archaea) as well as viruses. These trillions of microorganisms function like an “invisible organ” by helping in energy harvest and storage, contributing to metabolic functions, protecting humans against pathogens, and educating the immune system. Most studies target the predominant bacterial community of this “invisible organ” and the (so far detected) not-pathogenic archaeal community and its diversity remains largely hidden. The identification of archaea in human samples is hindered due to insufficient archaeal DNA amount in comparison with bacterial DNA and in particular the use of universal primers for Next Generation Sequencing (NGS) that have a low efficiency for targeting archaea. In this study, we aim to test the efficiency of several primer pairs specific for detecting archaeal taxa and diversity in human samples using the NGS approach and to assess the optimal workflow for data processing using three different publicly available pipelines (QIIME, mothur, and dada2). Here, we will present the data obtained from different human samples (stool, biopsies and skin) with two different archaeal specific primers and analyzed using three different workflows. Our results significantly contribute in understanding the meaning of Archaea in the human body.



17

P5 Assessing the resistance potential of microorganisms from archived samples of the International Space Station

Maximilian Mora, Alexandra Perras, Tatiana Alekhova, Robert Krause, Christine Moissl-Eichinger Medical University of Graz, Graz, Austria The International Space Station ISS is a unique and extreme biotope which is continuously inhabited by humans and their accompanying microbes since 2000. Almost complete isolation from any other environment and harsh environmental conditions, such as microgravity and enhanced background radiation, render the ISS a very special biotope and also the best model to assess microbial population dynamics during prolonged spaceflight. Assessing dynamics and also detecting resistances of the microbial population in this closed system is of utter importance for the planning of future long-term space flight missions. This study is part of the ARBEX project (ARchaeal and Bacterial EXtremophiles onboard the International Space Station ISS), which was designed to focus on the hardy, extremophilic bacteria and archaea, that have managed to adapt to the extreme conditions within the ISS modules. We analysed 8-12 years old dust samples from Russian ISS modules. The cultivable microbial diversity of these samples was assessed via a broad range of cultivation assays and the extremotolerant potential of the obtained isolates against desiccation, heat-shock and clinically relevant antibiotics was investigated. Additionaly, the total microbial signatures of these dust samples were assessed via Next Generation Sequencing. Besides a majority of human associated bacteria, we could also detect archaeal signatures as well as several microorganisms with a high resistance potential against physical and antibiotic stresses.



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P6 Expression and Interaction Studies of the Transfer Proteins TraN and TraK from the Gram-positive Conjugative Model Plasmid pIP501

Melanie Müller, Verena Kohler, Walter Keller Institute of Molecular Biosciences, University of Graz, Graz, Austria Conjugative transfer of plasmid encoded antibiotic resistance and pathogenicity genes constitutes a serious threat for hospitalized and immunosuppressed patients. During conjugation DNA is transferred from a donor to a recipient. The transfer is mediated by a plasmid encoded multi-protein complex, called Type IV Secretion System (T4SS). It is well studied in gram-negative bacteria, such as Escherichia coli, but scarce information is available for gram-positives (G+). Hence, we study the broad host range model plasmid pIP501 in Enterococcus faecalis. pIP501 encodes 15 putative transfer proteins (Tra-proteins), some of them have a high similarity to the virB system in Agrobacterium tumefaciens. So far, we could determine the structures of TraN and TraK among others. TraK has been shown to be essential for conjugative DNA transfer but the exact function of TraK in this process is unknown. We suspect that TraN has a function as repressor, but unfortunately we do not know anything about putative interaction partners. In this study we use quantitative real time PCR to find out more about the mRNA expression levels of TraN and TraK. We want to investigate if knockout mutants of TraN and TraK influence expression levels of other Tra-proteins. Additionally, we perform in vivo crosslinking experiments in Enterococcus faecalis to elucidate possible interaction partners of TraN and Trak. To identify the crosslinks analysis using mass spectrometry approaches will be undertaken. Complementary bacterial two hybrid (B2H) is performed.



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P7 Experimental evolution of polysaccharide-degrading Bacteroides thetaiotaomicron, an abundant member of the human gut

Nika Ivanovova, Christos Zioutis, Fatima Pereira, Madeleine Wyss, Andrew J. Macpherson, Kathy D. McCoy, David Berry University of Vienna, Department of Microbial Ecology and Ecosystem Science, Vienna, Austria The intestinal microbiota is important for human health and nutrition by degradation of recalcitrant compounds and synthesis of vitamins among many other host-beneficial activities. The genus Bacteroides is an abundant and diverse group that is associated with a healthy digestive tract. Bacteroides species are particularly adapted to utilization of complex dietary and host-derived compounds via an extensive repertoire of polysaccharide utilisation loci. These are evidence of genomic adaptation to survive in response to availability of various nutrient sources. However, the relationship between metabolic flexibility and fine-scale diversification and adaptation to different nutrient-based niches by Bacteroides species has not been extensively studied. Here, we focus on in vitro and in vivo evolution of one of the most broadly-studied species of this genus, Bacteroides thetaiotaomicron VPI-5482. Using antibiotic resistance markers in isogenic populations, we were able to track soft evolutionary sweeps in the first four weeks of colonization of germ-free mice, which revealed accumulation of single nucleotide polymorphisms as well as structural variations. Several polymorphisms appeared independently in multiple mice, indicating that there is a reproducible process of rapid genetic diversification in the murine gut that may be due to fitness selection. We are further studying the ability of B. thetaiotaomicron to digest different carbohydrates in vitro. Pure cultures were grown in defined minimal medium (Varel&Bryant, 1974) of single compounds as well as mixtures of carbohydrates such as amylopectin, pectin, and inulin. We believe that both in vitro and in vivo experimental evolution studies will identify novel colonization factors and to unravel the interplay of ecology and evolution in the assembly and function of the gut microbiota.



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P8 Antimicrobial volatile organic compounds: Identification of intra-cellular targets

Pascal Mülner, Laura Lange, Tomislav Cernava, Stefan Liebminger, Sepp D. Kohlwein, and Gabriele Berg ACIB GmbH, Graz, Austria In the past years volatile organic compounds (VOCs) from microbial origin have received increased attention. Volatile organic compounds play important roles in microbial signaling and biological control strategies to protect plants from diseases. Therefore they present an enormous potential for their use in industrial applications such as food processing. We have screened specific natural habitats with high occurrence of bacterial and fungal plant pathogens for disease-suppressing antagonists. These antagonists were shown to produce a variety of VOCs that inhibited the growth of a wide range of plant and human pathogens. Several highly bioactive diazine derivatives were found among the identified substances. Further studies confirmed the applicability of these substances in the treatment of fungal contaminants in the food processing industry and are currently evaluated as potential additives. The screening of an E.coli Keio Knockout Collection implies that the key component for their effectiveness seems to be membrane permeability. Furthermore, using headspace SPME GC-MS microbial interspecies communication was monitored where specific VOCs were identified which represent response signals to the emission of diazine derivatives. The current findings suggest that microbial VOCs can drastically shape the surrounding microbial environment and that these effects can be efficiently exploited for a variety of biotechnological as well as industrial applications.

P9 Community changes during the decontamination of hatching eggs using diazine derivates

Peter Kusstatscher, Tomislav Cernava, Stefan Liebminger, Gabriele Berg Institute of Environmental Biotechnology, Graz, Austria Microbial interplay relies on communication molecules that are exchanged between interacting microbes. Diazine derivatives among other volatile organic compounds (VOCs) were recently shown to play an important role in microbial interactions. The underlying effects were recognized to be useful for stabilizing applications in food processing. Currently, several approaches of egg decontamination are used in the hatching industry. The most frequently used method is formaldehyde fumigation. Formaldehyde is known to be a carcinogenic hazard and many hatcheries are searching for formaldehyde-free and environmental friendly methods. We evaluated the application of diazines on contaminated egg shells and found that they can drastically reduce the bacterial count. Moreover, we demonstrated that low diazine concentrations lead to changes in the microbial community structure due to their varying anti-microbial effects on different organisms. These changes were visualized with amplicon sequencing and complementing techniques to further optimize application strategies.



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P10 Genus Lactobacillus and subcutaneous adipose tissue thickness in women

Mörkl, S., Lackner, S., Müller, W., Gorkiewicz, G., Kashofer, K., Oberascher, A., Amouzadeh-Ghadikolai, O., Holasek, S.J. Medical University of Graz, Graz, Austria OBJECTIVE: In animal and human studies the genus Lactobacillus was shown to induce and promote weight gain. However, it is unclear whether or how Lactobacillus abundance affects body fat amount and distribution in women. METHODS: We have investigated the composition of the gut microbiome in a large cohort of 106 female participants: Anorexia nervosa patients (n=18), athletes (n=20), normal weight (n=26), overweight (n=22) and obese women (n=20). DNA was extracted from stool samples and subjected to 16s rRNA gene analysis. QIIME (a comprehensive bioinformatics pipeline) was used to analyze data. Additionally, we performed ultrasound measurements of subcutaneous adipose tissue thickness on specific body sites1. RESULTS: Kruskal-Wallis-Test was performed on relative genus Lactobacillus number of reads and showed significant differences between groups (H(4)=11,553, p=0,009). Mann-Whitney-U-tests identified that obese women had a significantly higher number of reads than anorexia nervosa patients, normal weight participants and athletes. BMI was positively correlated with Lactobacillus abundance (r=0,301, p=0,002). Ultrasound measurements of SAT-layer thickness at 8 different body sites as well as total SAT layer thickness showed significant positive correlations with lactobacillus number of reads. CONCLUSION: Lactobacillus genus correlates with SAT distribution in women. The comparatively high value of genus Lactobacillus found in athletes may indicate that Lactobacillus plays a role when high energy turnover is required. Further studies are needed to generalize these findings. References: 1 Müller, W. et al. (2016). Subcutaneous fat patterning in athletes: selection of appropriate sites and standardisation of a novel ultrasound measurement technique: ad hoc working group on body composition, health and performance, under the auspices of the IOC Medical Commission. British journal of sports medicine, 50(1), 45-54. Acknowledgement: Josef Smolle (Initiator of the ESAN-Study IC3490), Medical University Graz



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P11 Polyesterases from moss-assotiated microorganisms

Veronika Perz, Felice Quartinello, Christoph Provasnek, Georg Guebitz, Gabriele Berg, Christina A. Mueller Acib GmbH, Institut of Environmental Biotechnology - Boku Vienna, Tulln, Austria Plant-associated microorganisms are an interesting and potent source for new bioactive substances. Since the demand for enzymes with activities on synthetic substrates including man-made polymers increases continuously, we investigated a moss metagenome in terms of enzymatic activities. In detail, we surveyed the existence of polyesterases in microbial communities associated with Sphagnum magellanicum moss that is one key species within bog ecosystems. During the course of this study we identified six novel esterases that were heterologously expressed in Escherichia coli and were able to hydrolyze the synthetic copolyester poly(butylene adipate-co-butylene terephthalate) (PBAT). The two most promising enzymes EstB3 and EstC7 clustered into the family VIII of bacterial lipolytic enzymes, were purified and characterized using p-nitrophenyl butyrate (pHopt = 7.0 and 8.5, Topt = 48 °C and 50 °C, KM = 46.5 and 3.4 µM, respectively). This study underlines the high potential of plant-associated microbiomes for the discovery of novel biocatalysts that are even capable to hydrolyze synthetic polymers.



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P12 Effect of UV-R on skin microbiome and its interference in UV-induced immune suppression

VijayKumar Patra, Bettina Halwachs, Gregor Gorkiewicz and Peter Wolf Department of Dermatology, Medical University of Graz, Graz, Austria Ultraviolet-radiation (UV-R) is long known to have an intense effect on skin and its components, leading to activation of the innate immune system and ultimately suppressing the adaptive immune response in healthy individuals. This modulation of the immune system can be both beneficial and harmful. The skin hosts inumerable number of bacteria, fungi, viruses, arcahea and mites, and to date very little is known about the effects of UV-R on the skin microbiome. In this study we looked at the effect of different single doses of UV-B on the skin microbiome of the mouse at different time points and performed qPCR to determine expression levels of various antimicrobial peptides (AMPs). We further employed the model of induction of contact hypersensitivity (CHS) on disinfected (with local antiseptics) and germ-free mice to study interference of the microbiome in UV-induced immune suppression. We observed increased number of species at lower dose and reduced number at higher dose compared to control (UV-unexposed) mice. Interestingly, beta diversity analysis showed significant variation in microbial abundance at different doses and time points. We further performed LDA effect size (LefSe) analysis and observed abundance at taxonomic levels. qPCR for various AMPs showed differential expression levels suggesting that UV-B profoundly modulated the skin microbial load either directly or via expression of AMPs and affected its recolonization potential. Intriguingly, germ-free mice showed much higher levels of suppression of CHS (71%) compared to SPF mice (41%). A similar trend of more suppression of CHS was observed in disinfected mice compared to control mice. Collectively, our results suggest that the skin microbiome is sensitive towards UV-R and profoundly influences UV-induced immune suppression.



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P13 Investigation of the possible link between genetic background, gut microbiota and metabolism using the AJ/C57 chromosome substitution strains (CSSs)

Zahra. Safari1, Karl. Kashofer1, Christine. Moissl-Eichinger2, Gerald. Höfler1, Kurt. Zatloukal1* 1 Institute of Pathology Medical University Graz, Austria 2 Department of Internal Medicine, Medical University of Graz, Austria Steatosis is a major health problem worldwide, associated with diet, genetics and microbial factors. Previous data from our laboratory suggested that some of the specific chromosome substitution strains (CSSs), harboring one pair of chromosome from AJ in a B6 background, develop different phenotypes of steatosis under high-fat diet (HFD). To dissect this difference and study the contribution of the microbiome in steatosis development, 4 cohorts of CSSs with extreme phenotypes i.e., high and low level of steatosis were selected. They harbor one pair of chromosome 1, 8, 10 and 18 individually from AJ. Liver tissue as well as colon content for 16s rRNA analysis, were collected from 8 week HFD fed CSSs and founder strains. Our results showed notably high and low levels of fat deposition in the livers of CSS1 and CSS18 vs. CSS8 and CSS10 mice, respectively. This also correlated to a different microbiome composition of all the studied strains compared to that of the founders. Interestingly, growth of A. muciniphila was markedly higher in steatosis-resistant AJ strain, in variable amounts in CSSs, but rarely observed in steatosis-susceptible B6 strain possibly due to metabolic changes favoring its growth directed by one chromosome. Furthermore, the expression of some key genes involved in the fat metabolism pathway showed different patterns in different CSSs compared to the host strain, despite the similar liver morphology, suggesting the involvement of other underlying factors. Transcriptome and metabolome studies on liver tissue of all these strains will further elucidate the mechanisms. Overally, our results indicate the microbiome and genetic background as two independent factors influencing the response to steatosis in HFD treated CSSs.



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P14 Effects of probiotic therapy on the microbiota and mucosal immune system in patients with diarrhea-predominant irritable bowel syndrome

Adrian Mathias Moser1,2, Bettina Halwachs2,3,4, Walter Spindelboeck1,2, Heimo Strohmaier5, Patrizia Kump1,2, Gregor Gorkiewicz2,3,4, Christoph Hoegenauer1,2,4

1 Department of Internal Medicine, Division of Gastroenterology and Hepatology, Medical University Graz, Austria; 2 Theodor Escherich Laboratory for Microbiome Research, Medical University of Graz, 3Institute of Pathology, Medical University of Graz, Auenbruggerplatz 25, 8036 Graz, Austria 4 BioTechMed-Graz, Interuniversity Cooperation, Graz, Austria

5Center for Medical Research Graz (ZMF), Medical University Graz, Austria Background: The gut flora is suspected to be crucial for the development of diarrhoea-predominant irritable bowel syndrome (IBS-D). Although probiotics improve symptoms in IBS patients, the mechanisms of action are poorly understood. We investigated therefore the effect of probiotics in different regions of the gastrointestinal (GI)-tract. Methods: 10 patients with IBS-D were treated with a probiotic mixture for 4 weeks. Endoscopy was performed to collect gastric, duodenal and colonic specimens before and after treatment. We analysed dendritic cell (DC) and T cell subsets using FACS analysis of mucosal specimens. Stool samples were analyzed for zonulin and short-chain fatty acids (SCFA) content. Microbiota analysis was performed by 16s rRNA analysis in fecal and mucosal samples. Results: Changes in the microbiota after probiotic treatment varied between different regions of the GI tract. While diversity and richness were significantly increased in the stomach and duodenum this was not observed in the colon and in the stool. Bacteria from the probiotic could only be detected in stool samples by a slightly increased abundance of Lactobacillaceae. In stool samples zonulin concentrations were significantly lowered and SCFA concentrations increased by treatment. CD4+ T helper cells in the right-sided colon were reduced after probiotic treatment and a trend towards the induction of CD 4,8 double-negative T cells could be observed. Conclusion: Probiotic treatment in patients with IBS-D showed effects on mucosal immune cells, intestinal barrier function, short-chain fatty acid concentration, and the gastrointestinal microbiota. The effects varied considerably between regions of the GI tract.



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P15 Adaptation of Escherichia coli and Staphylococcus aureus to the natural compounds carvacrol and p-cymene and its impact on their antimicrobial susceptibility

Šimunović K., Klanćnik A., Janosity A., Tušek Žnidari? M., Bucar F., Kisko G., Smole Možina S. Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia Carvacrol and p-cymene are phenolic compounds found in essential oils from various plants, including oregano, thyme, lavender and others. These two compounds have been tested against many different bacteria and yeast, such as Salmonella spp., C. jejuni, E. coli, S. aureus and C. albicans and have proven to be efficient antimicrobials. With all antimicrobial substances, regardless of their origin and efficiency, there remains the question of microorganisms’ adaptation to them and the effect on susceptibility to other antimicrobials. In our research we adapted Escherichia coli and Staphylococcus aureus to 3 mM of carvacrol and 24 mM of p-cymene, with the MIC of non-adapted cultures being 1 and 8 mM, respectively. We compared the adapted and non-adapted cultures by investigating their susceptibility to 4 antibiotics (erythromycin, ciprofloxacin, dirithromycin, and rifampicin) with the microdilution method, their membrane integrity with the LIVE/DEAD® BacLightTM bacterial viability kit and by examining the effect on the bacteria’s efflux pumps with the ethidium bromide accumulation method. Our results show a stable adaptation of E. coli to p-cymene but not to carvacrol, and of S. aureus to both compounds. The membrane integrity of all adapted bacteria was significantly lower compared to the non-adapted culture. The ethidium bromide accumulation of adapted S. aureus was significantly lower, indicating overactive efflux pumps of the adapted culture, whereas the ethidium bromide accumulation in adapted E. coli had no visible changes. The most important change was seen in the increased susceptibility of the adapted cultures to some antibiotics.

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Abstracts of the - Med Uni Graz ESCHERICH SYMPOSIUM 2016: ABSTRACTS Table of Contents 4 P14 Effects of probiotic therapy on the microbiota and mucosal immune system in patients with